Female-specific neuroprotection against transient brain ischemia observed in mice devoid of prion protein is abolished by ectopic expression of prion protein-like protein.

This study was designed to examine the function of cellular prion protein and prion protein-like protein/Doppel, in transient ischemia-related neuronal death in the hippocampus. Two different lines of mice devoid of cellular prion protein, Zrch I Prnp(0/0) and Ngsk Prnp(0/0), were used. The former lacks cellular prion protein whereas the latter ectopically expresses prion protein-like protein/Doppel in the brain in the absence of cellular prion protein. Mice were subjected to 10 min-occlusion of the bilateral common carotid arteries with recovery for 14 days. Less than 10% of the pyramidal neurons in the CA1 subfield were degenerated in male and female wild-type mice. In contrast, more than half of the neurons were lost in male Zrch I Prnp(0/0) and Ngsk Prnp(0/0) mice. Such severe neuronal loss was also observed in female Ngsk Prnp(0/0) mice. However, female Zrch I Prnp(0/0) mice showed mild neuronal loss similar to wild-type mice. Flunarizine, a T- and L-type Ca(2+)-channel antagonist, significantly reduced the neuronal loss in female but not in male Ngsk Prnp(0/0) mice. These results indicate that loss of cellular prion protein renders hippocampal neurons susceptible to ischemic insult specifically in male but not female mice and the ectopic expression of prion protein-like protein/Doppel aggravates the ischemic neuronal death in female prion protein-null mice probably via overloading of Ca(2+)-dependent signaling.

HTLV-1 proviruses encoding non-functional TAX in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1). TAX, the major transactivator of HTLV-1, has been implicated in the immortalization of infected T-cells, but molecular mechanisms of in vivo malignant cell transformation induced by HTLV-1 remain unclear. To investigate the role of TAX in the monoclonal proliferation of ATL cells, we determined the nucleotide sequence of tax DNA clones obtained from 6 ATL patients and analysed the biological function of their products. We found that ATL cells from 2 of these patients possessed tax with a nonsense or frame-shift mutation resulting in the premature termination of its protein product, which was no longer functional. This strongly argued against an indispensable role of TAX for the maintenance of ATL cells in vivo. On the other hand, the frequency of nucleotide substitutions found in non-functional tax DNA clones from these patients was significantly lower than those in functional tax DNA clones from the others, suggesting a role for TAX in the genome instability of infected cells. Although mismatch repair defects in the microsatellite markers, including those in hMSH3, hMSH6, BAX, TGF-beta RII, and E2F4 genes, were infrequent, we found an increase in the number of CAG repeats of the E2F4 microsatellite marker in 1 patient. These findings indicate that while TAX may be a necessary prerequisite for malignant transformation of infected cells, it is not essential for the maintenance of ATL cells in vivo.

Abnormal activation of glial cells in the brains of prion protein-deficient mice ectopically expressing prion protein-like protein, PrPLP/Dpl.

BACKGROUND: Some lines of mice homozygous for a disrupted prion protein gene (Prnp), including Ngsk Prnp(0/0) mice, exhibit Purkinje cell degeneration as a consequence of the ectopic overexpression of the downstream gene for prion protein-like protein (PrPLP/Dpl) in the brain, but others, such as Zrch I Prnp(0/0) mice, show neither the neurodegeneration nor the expression of PrPLP/Dpl. In the present study, we found that Ngsk Prnp(0/0), but not Zrch I Prnp(0/0) mice, developed gliosis involving both astrocytes and microglia in the brain. MATERIALS AND METHODS: The brains from wild-type (Prnp(+/+)), Ngsk Prnp(0/0), Zrch I Prnp(0/0), and reconstituted Ngsk Prnp(0/0) mice carrying a mouse PrP transgene, designated Tg(P) Ngsk Prnp(0/0) mice, were subjected into Northern blotting and in situ hybridization using probes of glial fibrillary acidic protein (GFAP) and lysozyme M (LM) specific for astrocytes and microglia, respectively. Immunohistochemistry was also performed on the brain sections using anti-GFAP and anti-F4/80 antibodies. RESULTS: Northern blotting demonstrated upregulated expression of the genes for GFAP and LM in the brains of Ngsk Prnp(0/0), but not in Zrch I Prnp(0/0) mice. A transgene for normal mouse PrP(C) successfully rescued Ngsk Prnp(0/0) mice from the glial activation. In situ hybridization and immunohistochemistry revealed activated astrocytes and microglia mainly in the white matter of both the forebrains and cerebella. In contrast, there was no evidence of neuronal injury except for the Purkinje cell degeneration. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. CONCLUSIONS: These findings strongly suggest that ectopic PrPLP/Dpl in the absence of PrP(C) is actively involved in the glial-cell activation in the brain.

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C).

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.

Early appearance but lagged accumulation of detergent-insoluble prion protein in the brains of mice inoculated with a mouse-adapted Creutzfeldt-Jakob disease agent.

1. To elucidate mechanisms for the generation of the detergent-insoluble, proteinase K-resistant prion protein (PrP(Sc)) from the detergent-soluble, proteinase K-sensitive PrP (PrP(C)) and the replication of the infectious agent in prion diseases, we followed the kinetics of detergent-insoluble PrP and PrP(Sc) levels, infectious titers, and associated pathological changes in the brains of mice inoculated with a mouse-adapted Creutzfeldt Jakob disease agent. 2. PrP(Sc) in brain homogenate and detergent-insoluble PrP enriched by two-cycle ultracentrifugation were detected by immunoblotting and their relative amounts were estimated according to a standard curve plotted between the amount of PrP and signal intensity on immunoblotting. The titer of infectivity was determined by the incubation periods of mice inoculated with the unfractionated homogenate on the basis of a standard curve plotted between the titer and incubation period. 3. Detergent-insoluble PrP became detectable 4 weeks postinoculation (p.i.) well before the detection of PrP(Sc). The low level of detergent-insoluble PrP continued until dramatic accumulation occurred at 14 weeks p.i., correlating well with the accumulation of PrP(Sc) and development of pathological changes. The infectious titer was undetectable at 4 weeks p.i. and its logarithmic increase occurred 10 weeks p.i. preceding the logarithmic accumulation of PrPs. 4. The lag time of detergent-insoluble PrP accumulation and the discrepancy between infectious titers and PrPs observed during the early period after inoculation suggest a slow and rate-limiting step for the detergent-insoluble PrP to become the infectious agent-associated PrP(Sc).

Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

Identification of genes associated with the progression of adult T cell leukemia (ATL).

Patients with adult T-cell leukemia/lymphoma (ATL) exhibit a variety of clinical features, and this disease is therefore clinically subclassified into acute, lymphomatous, chronic, and smoldering types. Acute ATL is a typical leukemic form of ATL with rapid progression, and chronic ATL is a less aggressive clinical form allowing long-term survival even without chemotherapy. In the present study, we used fresh peripheral blood mononuclear cells (PBMC) from both types of ATL patients to identify molecules that may contribute to the difference between acute and chronic ATL. Isolated mRNAs expressed differentially between the two types of ATL include a T-cell differentiation antigen (MAL), a lymphoid-specific member of the G-protein-coupled receptor family (EBI-1 / CCR7), a novel human homologue to a subunit (MNLL) of the bovine ubiquinone oxidoreductase complex, and a human fibrinogen-like protein (hpT49). We found that the former three are upregulated in acute ATL and the last is down-regulated in both chronic and acute ATL. We speculate that dysregulation of the genes may account for the malignant features of ATL cells, in terms of growth, energy metabolism, and motility.

Prion infection impairs the cellular response to oxidative stress.

The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies remains uncertain. In this study, it was demonstrated that prion-infected hypothalamic neuronal GT1 cells displayed a higher sensitivity to induced oxidative stress over noninfected cells. In addition, the infected cells presented an increased lipid peroxidation and signs of apoptosis associated with a dramatic reduction in the activities of the glutathione-dependent and superoxide dismutase antioxidant systems. This study indicates for the first time that prion infection results in an alteration of the molecular mechanisms promoting cellular resistance to reactive oxygen species. This finding is vital for future therapeutic approaches in transmissible spongiform encephalopathies and the understanding of the function of the prion protein.

Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration.

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Comparative molecular analysis of HTLV-I proviral DNA in HTLV-I infected members of a family with a discordant HTLV-I-associated myelopathy in monozygotic twins.

In order to elucidate the underlying mechanisms of a discordant case with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in monozygotic twins, we investigated HTLV-I tax sequences of 10 - 18 polymerase chain reaction-based clones each derived from peripheral blood mononuclear cells of the twins as well as their infected mother and an elder brother who also suffered from HAM/TSP. Sequence comparison revealed that three of the infected individuals including a twin with HAM/TSP shared the consensus tax sequence identical to the reference, ATK-1, but that of another healthy twin was different at five nucleotide positions including three nonsynonymous changes from ATK-1. This finding strongly suggested that different HTLV-I strains infected the monozygotic twins and the difference in infected proviral sequences determined the discordant clinical outcomes. Transfection and subsequent reporter assays failed to show a significant difference in transactivation activity on HTLV-I LTR and NF-kappaB elements between the products of the two sequences. Two HAM/TSP patients (a twin and elder brother) among three members infected with the ATK-1 type virus shared a paternal HLA allele which was absent in the healthy individual (mother). Genetic analysis of sequence variation in the tax sequences of the discordant twins showed that the Dn/Ds ratio was high in the healthy twin but low in the twin with HAM/TSP, implying the presence of more intense selection forces in the carrier. Our findings strongly suggested that a particular combination of HTLV-I strains with an HLA genotype would be a risk for HAM/TSP.

[Physiopathology and molecular diagnosis for prion diseases].

Prion diseases, including Creutzfeldt-Jakob disease (CJD), are infectious neurodegenerative disorders. The etiological agent, prion, is postulated to consist mainly of a proteinase K-resistant isoform of prion protein (PrPSc) which is generated by post-translational conversion from the proteinase K-sensitive normal version (PrPC) physiologically expressed on the surface of neuronal and glial cells. The constitutive conversion results in the tremendous accumulation of PrPSc in the prion-infected brain. Homozygous disruption of the Prnp gene encoding PrPC renders mice resistant to prion, and the animals are no longer capable of generating PrPSc, indicating an essential role for PrPSc in the pathogenesis of prion diseases. The PrP-null mice (Ngsk Prnp0/0) revealed progressive ataxia due to the degeneration of cellebellar Purkinje cells at old ages. Successful rescue of Ngsk Prnp0/0 mice from neurodegeneration by a transgene encoding the normal mouse PrPC has indicated that the functional loss of PrPC is essential for this phenotype. Moreover, we detected aberrant mRNAs chimeric between Prnp exon 1-2 and a novel gene encoding PrP-like protein (PrPLP). These results suggested that, in addition to the functional loss of PrPC, ectopic expression of the PrPLP in the brain of Ngsk Prnp0/0 mice could be associated with Purkinje cell degeneration.

Gene expression profile in prion protein-deficient fibroblasts in culture.

To investigate the physiological function of the cellular isoform of prion protein (PrP(C)), the gene expression profile was studied by analyzing a cDNA expression array containing 597 clones of various functional classes in two distinct skin fibroblast cell lines designated SFK and SFH, established from PrP-deficient (PrP(-)(/-)) mice and PrP(+/+) mice, respectively. The cells were incubated in the culture medium with or without inclusion of basic fibroblast growth factor (bFGF). When SFK cells were compared with SFH cells in untreated conditions, the expression of 15 genes, including those essential for cell proliferation and adhesion, was reduced, whereas the expression of 27 genes, including those involved in the insulin-like growth factor-I (IGF-I) signaling pathway, was elevated. Northern blot analysis verified a significant down-regulation of the receptor tyrosine kinase substrate Eps8, cyclin D1, and CD44 mRNAs, and a substantial up-regulation of phosphatidylinositol 3-kinase p85, IGF-I, and serine protease inhibitor-2.2 mRNAs in SFK cells. The patterns of induction or reduction of gene expression after exposure to bFGF showed considerable overlap between both cell types. Furthermore, both Eps8 and CD44 mRNA levels were reduced greatly in the brain tissues of the cerebrum isolated from the PrP(-)(/-) mice. These results indicate that the disruption of the PrP gene resulted in an aberrant regulation of a battery of genes important for cell proliferation, differentiation, and survival, including those located in the Ras and Rac signaling pathways.

Up-regulation of human T lymphotropic virus type 1 (HTLV-1) tax/rex mRNA in infected lung tissues.

HTLV-1 has been implicated in certain pulmonary diseases. We previously demonstrated that expression of HTLV-1 tax/rex mRNA, encoding the transcriptional transactivator Tax, was closely associated with infiltration of activated T lymphocytes into lung tissue. To explore mechanisms of tax/rex expression in the lung, tax/rex mRNA expression and proviral DNA load were compared between peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) from four patients with HTLV-1-associated myelopathy (HAM/TSP) and 13 carriers with various pulmonary symptoms. Semiquantitative detection of tax/rex mRNA strongly suggested that the lung was a preferential site for its expression. Proviral DNA loads in non-HAM/TSP carriers were variable but correlated well between PBMC and BALC in each individual, and revealed no relationship with tax/rex mRNA expression. In contrast, both cell groups from four HAM/TSP patients expressed detectable tax/rex mRNA accompanied by high proviral DNA load. The ratio of tax/rex mRNA expression to proviral DNA load was higher in BALC than in PBMC in three of four carriers and in three of four HAM/TSP patients, suggesting up-regulation of tax/rex mRNA in infected lung tissue. To analyse differences in distribution of HTLV-1 quasispecies between the two tissues, phylogenetic analysis was performed for sequence sets of the proviral tax open reading frame (ORF: 1059 bp) derived from PBMC and BALC of two infected individuals. Sequences derived from the two tissues distributed similarly to branches of phylogenetic trees, and there was no evidence of selective distribution of certain quasispecies in the lung. Our results suggest the presence of tissue-specific conditions that activate viral expression in infected cells in the lung. Constitutive exposure of this tissue to foreign antigens leading to up-regulation of basal viral promoter activity is likely to be one such mechanism.

High frequency of postnatal transmission of TT virus in infancy.

DNA of TT virus (TTV), a novel human circovirus, was tested for in 116 mother-infant pairs who had participated in the adult T-cell leukemia prevention program (APP) in Nagasaki, Japan, and refrained from breast-feeding. By polymerase chain reaction with Okamoto's seminested primers, 36 of the 115 (31%) mothers were positive. At the age of 6-8 months, 7 of 29 (24%) and 6 of 72 (8%) infants born to infected and uninfected mothers were positive, respectively (P = 0.047; RR, 2.90). Maternal TTV DNA load did not correlate with infantile infections. Since 99 of 100 (99%) cord blood samples were negative and all the mothers refrained from breast-feeding, the infantile TTV transmission would not be intrauterine or milk-borne. Between 6-8 and 12-21 months of age, 4 of 12 (33%) and 5 of 22 (23%) children born to infected and uninfected mothers turned positive, respectively (NS). At 12-21 months of age, 8 of 21 (38%) and 12 of 32 (38%) children born to infected and uninfected mothers were positive, respectively (NS). These results indicate that the TTV infection prevails in children at a frequency comparative to that in their mothers within the first 2 years of life, regardless of the maternal TTV status.

Demographic and reproductive factors for high seroprevalence of Chlamydia trachomatis among pregnant women in Japan.

In order to elucidate demographic and reproductive factors associated with Chlamydia trachomatis seropositivity, serological screening and questionnaire survey were conducted on pregnant women in Nagasaki Prefecture, Japan. Serum samples were taken from 1718 pregnant women between September and December, 1996, at the cooperative obstetric hospitals and clinics, and tested for the presence of antibodies to C. trachomatis using the enzyme immunoassay. A questionnaire was administered on a sub-sample (n -409), among whom 85 (20.8%) were seropositive. A multiple logistic analysis revealed that four characteristics showed a significant association with the seropositivity: (i) experience of premarital pregnancy, (ii) non use of condoms, (iii) short duration of education, and (iv) more frequent induced abortion. The unsafe sexual behavior of young people lacking proper knowledge of how to prevent STD is the most important intervention target for control of the C. trachomatis epidemic in Japan.

Evaluation of adult T-cell leukemia/lymphoma incidence and its impact on non-Hodgkin lymphoma incidence in southwestern Japan.

The incidence of adult T-cell leukemia/lymphoma (ATL) and its impact on that of total non-Hodgkin lymphoma (NHL) were evaluated in Nagasaki, an area in southwestern Japan where human T-cell lymphotropic virus type I (HTLV-I) is endemic. The first study area comprised 4 towns located on the K Islands, which had a population of 26,870 in 1990. The overall HTLV-I seroprevalence estimated from the serologic survey of 18,485 subjects was 16.2%. By using the data from the Nagasaki Prefectural Cancer Registry (NPCR) and reviewing clinical and laboratory information, we identified 40 cases of ATL and 35 cases of other NHL diagnosed between 1985 and 1995. The crude annual incidence of ATL among 100,000 HTLV-I carriers aged 30 or older was estimated at 137.7 for men and 57.4 for women, with a significant sex difference after adjustment for age (rate ratio = 2.50, 95% confidence interval 1.32-4.73). The cumulative risk from 30 to 79 years of age was estimated at approximately 6.6% for men and 2.1% for women. Among the entire population, ATL accounted for 51 to 59% of the total NHL incidence, showing the strong impact of HTLV-I infection. The second study area comprised the whole of Nagasaki Prefecture (total population in 1990 = 1.56 million). Between 1985 and 1995, 989 cases of ATL and 1,745 cases of other NHL were registered in the NPCR. The world age-standardized annual incidence rate of ATL per 100,000 persons aged 30 or older was estimated at 10.5 for men and 6.0 for women, which accounted for approximately 37 to 41% of the total NHL incidence.

Upregulation of the genes encoding lysosomal hydrolases, a perforin-like protein, and peroxidases in the brains of mice affected with an experimental prion disease.

In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of beta-N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrP(Sc)) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

The novel locus Prnd is 16 kb downstream of the mouse prion protein (PrP) gene Prnp and encodes a 179 residue PrP-like protein designated doppel (Dpl). Prnd generates major transcripts of 1.7 and 2.7 kb as well as some unusual chimeric transcripts generated by intergenic splicing with Prnp. Like PrP, Dpl mRNA is expressed during embryogenesis but, in contrast to PrP, it is expressed minimally in the CNS. Unexpectedly, Dpl is upregulated in the CNS of two PrP-deficient (Prnp(0/0)) lines of mice, both of which develop late-onset ataxia, suggesting that Dpl may provoke neurodegeneration. Dpl is the first PrP-like protein to be described in mammals, and since Dpl seems to cause neurodegeneration similar to PrP, the linked expression of the Prnp and Prnd genes may play a previously unrecognized role in the pathogenesis of prion diseases or other illnesses.

A mouse prion protein transgene rescues mice deficient for the prion protein gene from purkinje cell degeneration and demyelination.

Disruption of both alleles of the prion protein gene, Prnp, renders mice resistant to prions; in a Prnp o/o line reported by some of us, mice progressively developed ataxia and Purkinje cell loss. Here we report torpedo-like axonal swellings associated with residual Purkinje cells in Prnp o/o mice, and we demonstrate abnormal myelination in the spinal cord and peripheral nerves in mice from two independently established Prnp o/o lines. Mice were successfully rescued from both demyelination and Purkinje cell degeneration by introduction of a transgene encoding wild-type mouse cellular prion protein. These findings suggest that cellular prion protein expression may be necessary to maintain the integrity of the nervous system.